Cilia and flagella are complex microtubular organelles that are ubiquitous among eukaryotes, including humans, where they are found in the respiratory tract, the female reproductive tract and on sperm cells. Ciliary and flagellar dysfunction resulting from the absence of dynein arms has been reported to cause respiratory and fertility problems in humans and other mammals. The long term objectives of this work are to understand the mechanisms by which dyneins assemble into ciliary axonemes and to understand the role(s) of dyneins in ciliary motility by using the unicellular protozoan, Tetrahymena thermophila. as a model system. Specifically, the role(s) that a newly discovered ciliary dynein heavy chain (sky chain dynein heavy chain) plays in ciliary motility will be determined by a) cloning, sequencing and analyzing the gene encoding the sky chain dynein heavy chain, b) introducing mutations into the sky chain dynein heavy chain gene using targeted gene knock out techniques, and c) analyzing the mutant strains to allow inferences to be drawn about the location and function of the sky chain dynein heavy chain. To clone the sky chain gene, the sky chain will be isolated and partially sequenced. The sequences will be used to design oligonucleotide primers. The primers will be used in the polymerase chain reaction to amplify DNA encoding a portion of the sky chain dynein heavy chain gene. To create the knockout vector, an expression cassette containing a selectable marker will be inserted into the middle of the sky chain gene. The knock out vector will be transformed into conjugating Tetrahymena under conditions that promote homologous recombination between the transforming DNA and the target sequence. This should yield mutant strains in which endogenous sky chain genes are inactivated by insertion of the expression cassette and thus, are missing the sky chain dynein heavy chain. Mutant strains will be subjected to biochemical and ultrastructural analyses to determine the location and function of the newly discovered sky chain dynein heavy chain. The results of this work should add to our understanding of dynein organization and function in cilia and flagella.